Mathematical model of influenza A virus production in large-scale microcarrier culture.

A mathematical model that describes the replication of influenza A virus in animal cells in large-scale microcarrier culture is presented. The virus is produced in a two-step process, which begins with the growth of adherent Madin-Darby canine kidney (MDCK) cells. After several washing steps serum-free virus maintenance medium is added, and the cells are infected with equine influenza virus (A/Equi 2 (H3N8), Newmarket 1/93). A time-delayed model is considered that has three state variables: the number of uninfected cells, infected cells, and free virus particles. It is assumed that uninfected cells adsorb the virus added at the time of infection. The infection rate is proportional to the number of uninfected cells and free virions. Depending on multiplicity of infection (MOI), not necessarily all cells are infected by this first step leading to the production of free virions. Newly produced viruses can infect the remaining uninfected cells in a chain reaction. To follow the time course of virus replication, infected cells were stained with fluorescent antibodies. Quantitation of influenza viruses by a hemagglutination assay (HA) enabled the estimation of the total number of new virions produced, which is relevant for the production of inactivated influenza vaccines. It takes about 4-6 h before visibly infected cells can be identified on the microcarriers followed by a strong increase in HA titers after 15-16 h in the medium. Maximum virus yield Vmax was about 1x10(10) virions/mL (2.4 log HA units/100 microL), which corresponds to a burst size ratio of about 18,755 virus particles produced per cell. The model tracks the time course of uninfected and infected cells as well as virus production. It suggests that small variations (<10%) in initial values and specific rates do not have a significant influence on Vmax. The main parameters relevant for the optimization of virus antigen yields are specific virus replication rate and specific cell death rate due to infection. Simulation studies indicate that a mathematical model that neglects the delay between virus infection and the release of new virions gives similar results with respect to overall virus dynamics compared with a time delayed model.

[1]  A. Osterhaus,et al.  Comparison of RNA hybridization, hemagglutination assay, titration of infectious virus and immunofluorescence as methods for monitoring influenza virus replication in vitro. , 1998, Journal of virological methods.

[2]  A S Perelson,et al.  Differences in viral dynamics between genotypes 1 and 2 of hepatitis C virus. , 2000, The Journal of infectious diseases.

[3]  M. Nowak,et al.  The dynamics of HTLV-I and the CTL response. , 1999, Immunology today.

[4]  G. Whittaker,et al.  Early stages of influenza virus entry into Mv-1 lung cells: involvement of dynamin. , 2000, Virology.

[5]  M. Betenbaugh,et al.  Life and death in mammalian cell culture: strategies for apoptosis inhibition. , 2004, Trends in biotechnology.

[6]  D. Endy,et al.  Intracellular kinetics of a growing virus: a genetically structured simulation for bacteriophage T7. , 1997, Biotechnology and bioengineering.

[7]  F. Dorner,et al.  Development of a mammalian cell (Vero) derived candidate influenza virus vaccine. , 1998, Vaccine.

[8]  A. Fooks,et al.  Comparison of large-scale mammalian cell culture systems with egg culture for the production of influenza virus A vaccine strains. , 2001, Vaccine.

[9]  M L Shuler,et al.  A mathematical model of the trafficking of acid-dependent enveloped viruses: application to the binding, uptake, and nuclear accumulation of baculovirus. , 1997, Biotechnology and bioengineering.

[10]  G. Neumann,et al.  Reverse genetics of influenza virus. , 2001, Virology.

[11]  S. Nir,et al.  Interactions of influenza virus with cultured cells: detailed kinetic modeling of binding and endocytosis. , 1999, Biochemistry.

[12]  J E Bailey,et al.  Modeling the population dynamics of baculovirus‐infected insect cells: Optimizing infection strategies for enhanced recombinant protein yields , 1992, Biotechnology and bioengineering.

[13]  M L Shuler,et al.  A model of the binding, entry, uncoating, and RNA synthesis of Semliki Forest virus in baby hamster kidney (BHK‐21) cells , 1995, Biotechnology and bioengineering.

[14]  Beda Joos,et al.  Quantification of in vivo replicative capacity of HIV-1 in different compartments of infected cells. , 2001 .

[15]  Graham F. Medley,et al.  Hepatitis-B virus endemicity: heterogeneity, catastrophic dynamics and control , 2001, Nature Medicine.

[16]  A. Perelson,et al.  Optimizing within-host viral fitness: infected cell lifespan and virion production rate. , 2004, Journal of theoretical biology.

[17]  J. Kehren,et al.  bcl-2 alters influenza virus yield, spread, and hemagglutinin glycosylation , 1996, Journal of virology.

[18]  M. Tabata,et al.  Analysis of the infection system of human T‐cell leukaemia virus type I based on a mathematical epidemic model , 2001, Statistics in medicine.

[19]  U. Reichl,et al.  Development of bioprocess concepts on vaccine production : influenza virus as an example , 2001 .

[20]  G. Neumann,et al.  Genetic engineering of influenza and other negative-strand RNA viruses containing segmented genomes. , 1999, Advances in virus research.

[21]  A. Osterhaus,et al.  Characterization of high-growth reassortant influenza A viruses generated in MDCK cells cultured in serum-free medium. , 1999, Vaccine.

[22]  U Reichl,et al.  Metabolism of MDCK cells during cell growth and influenza virus production in large-scale microcarrier culture. , 2004, Vaccine.

[23]  Z Xu,et al.  A mathematical model of hepatitis B virus transmission and its application for vaccination strategy in china. , 2000, International journal of epidemiology.

[24]  S. Pleschka,et al.  Negative-strand RNA viruses: genetic engineering and applications. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[25]  Patrick W Nelson,et al.  Mathematical analysis of delay differential equation models of HIV-1 infection. , 2002, Mathematical biosciences.

[26]  T. Parslow,et al.  Evidence for Segment-Nonspecific Packaging of the Influenza A Virus Genome , 2002, Journal of Virology.

[27]  C. D. de Gooijer,et al.  A structured dynamic model for the baculovirus infection process in insect‐cell reactor configurations , 1992, Biotechnology and bioengineering.

[28]  Morgan H. McCoy,et al.  Caspase activation in equine influenza virus induced apoptotic cell death. , 2002, Veterinary microbiology.

[29]  J. Yin,et al.  Quantitative intracellular kinetics of HIV type 1. , 1999, AIDS research and human retroviruses.

[30]  A. Attwell,et al.  Replicative advantage in tissue culture of egg-adapted influenza virus over tissue-culture derived virus: implications for vaccine manufacture. , 1995, Vaccine.

[31]  Achim Kienle,et al.  Object-oriented modeling of distillation processes , 1999 .

[32]  C. S. Sanderson,et al.  Structured modeling of recombinant protein production in batch and fed-batch culture of baculovirus-infected insect cells , 2004, Cytotechnology.

[33]  Martin H. Koldijk,et al.  The human cell line PER.C6 provides a new manufacturing system for the production of influenza vaccines. , 2001, Vaccine.

[34]  A. Garfinkel,et al.  Primary HIV infection of infants: the effects of somatic growth on lymphocyte and virus dynamics. , 1999, Clinical immunology.